Bispecific antibodies which recognize two different antigens, for example a tumor cell surface antigen and an effector molecule, are widely used in experimental immunotherapy (Fanger et al., Crit. Rev. Immunol. 12, 101-124 (1992); van de Winkel et al., Immunol. Today 18, 562-564 (1997)). The effector functions recruited by bispecific antibodies include those which occur naturally in the body, such as, cytotoxic and phagocytic cells, complement components, cytokines and thrombolytic and fibrinolytic enzymes, as well as exogenous effector molecules such as, toxins, prodrug-converting enzymes and radionuclides. Thus, for example, injection of bispecific antibodies against Hodgkin's tumor-associated antigen CD30 and the T-cell antigens CD3 and CD28 in xenotransplanted tumors leads to recruitment and stimulation of cytotoxic T cells and to induction of a tumoricidal activity (Renner et al., Science 264, 833, 1994).
In another approach, a bispecific antibody against CD30 and alkaline phosphatase was used to recruit the enzyme to the tumor site and thus to convert the nontoxic precursor of a drug into a toxic drug (Sahin et al., Cancer Res. 50, 6944-6948, 1990).
An alternative to injection of the purified bispecific antibody is expression and secretion of these bispecific antibodies by cells transfected in vitro or in vivo. The advantage of this strategy is that production of the bispecific antibody by the transduced cells takes place in vivo and thus there is no need for elaborate production and purification of the bispecific antibody before injection. In addition, it is possible by choosing suitable expression systems to control the expression of the bispecific antibody locally, in organs or a tumor or else systemically.
Bispecific antibodies can be prepared, for example, by chemical crosslinking (Nisonoff et al., Nature 194, 355, 1962). There are, however, a number of drawbacks associated with this approach. Upon chemical crosslinking of monoclonal or polyclonal antibody molecules of animal origin there may be inactivation of no small proportion. In addition, both hetero- and homodimers are produced. Homodimers must be separated from the required heterodimers by elaborate processes. Hybridoma cells which produce bispecific antibodies, called hybrid hybridomas, can be prepared only in a relatively elaborate way because it is necessary to fuse together two different hybridomas (Milstein et al., Nature 305, 537, 1983). The proportion of functional heterodimers is relatively low, theoretically only 10%, because the heavy and light chains of the two antibody molecules can associate with one another in any of a large number of ways. Furthermore, the starting material used mainly comprises murine monoclonal antibodies which, in turn, are immunogenic for humans.
Various bivalent or bispecific antibody molecules can also be prepared recombinantly and expressed in bacteria or eukaryotic cells (WO 93/06217). It is common to all recombinant antibody molecules that both murine and human starting molecules can be used to prepare them. Various methods have been developed to allow bispecific recombinant antibody molecules to be prepared as efficiently as possible. Various groups of molecules can be prepared using these methods.
In one group of molecules, the variable parts of the antibodies are fused to constant immunoglobulin domains (Fc, CH3, CL), in order to achieve dimerization (Hu et al., Cancer Res. 56, 3055-3061 (1994); Hayden et al., Ther. Immunol. 1, 3-15 (1994)). In this case, however, there is no selection for the desired heterodimeric molecules, so that mainly bivalent homodimers are produced in this way. Expression of these molecules in functional form is, moreover, confined to eukaryotic cells.
In another group of molecules, the variable parts of the antibodies are fused to peptides or protein domains from other proteins to prepare bivalent or bispecific molecules (Plückthun and Pack, Immunotechnol. 3, 83-105 (1997)). In this case too there is usually formation of homo- and heterodimers because of the random association. In addition, these molecules contain a proportion, which is considerable in some cases, of foreign sequences, so that a marked immunogenicity is to be expected.
In a third group of molecules, slight modifications of recombinant Fv fragments, usually single-chain Fv fragments (scFv), are used to prepare bivalent or bispecific molecules (Holliger and Winter, Curr. Opin. Biotechnol. 4, 446-449 (1993)). These include dimerization through additional cysteines at the C terminus of the scFv chains (MacCartney et al., Protein Engin. 8, 301-314 (1994)). However, this results in both homo- and heterodimers and, when expressed in bacterial cells, non-functional aggregates are produced. Tandem-scFv molecules of the structure scFv(A)-linker-scFv(B) (Mallender and Voss, J. Biol. Chem. 269, 199-206, 1994) can be expressed both in bacteria and in eukaryotic cells. However, in some of these cases non-functional associations of the four variable domains may also occur.
Recombinant antibody technology has led in recent years to the development of novel small, bivalent or bispecific antibody fragments. Examples of molecules of this type are the “diabodies” (Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993). These comprise variable VH and VL domains of immunoglobulins which are connected together by a very short linker. This linker is too short to bring about association of the VH and VL domains of the same chain, as occurs with single-chain Fv fragments. This means that the VH and VL domains of two chains associate to form a dimer, so that molecules with two binding sites are produced (Perisic et al., Structure 2, 1217-1226, 1994). Bispecific “diabodies” are produced by the expression of two chains of the structure VH(A)-VL(B) and VH(B)-VL(A) in one cell. In this case, VL means the variable (V) domain of the light (L) chain and VH means the variable domain (V) of the heavy (H) chain of an immunoglobulin, these variable domains binding the antigen (A) or (B). The association of the VH parts with the VL parts produces heterodimeric fragments with functionally active binding sites. Bacterially expressed bispecific “diabodies” have already been employed successfully for recruiting various effector molecules, immunoglobulins, C1q or enzymes or effector cells such as, for example, cytotoxic T lymphocytes (Kontermann et al., Nature Biotechnol. 15, 629 (1997); Holliger et al., Protein Engin. 9, 299 (1996); Nature Biotechnol. 15, 632 (1997), Zhu et al., BioTechnol. 14, 192 (1996); FitzGerald et al., Protein Engin. 10, 1221 (1997); Krebs et al., J. Biol. Chem. 273, 2858 (1998)).
“Diabodies”, however, also have several disadvantages. For example since the two VH(A)-VL(B) and VH(B)-VL(A) chains are no longer physically connected, homo- and heterodimers can be produced in equal amounts. This makes very elaborate purification processes necessary for the heterodimers. In addition, dissociation of the dimers occurs, as has already been shown for scFv fragments (Glockshuber et al., Biochem. 29, 1362-1367 (1990)). To solve this problem, disulfide-stabilized “diabodies” (FitzGerald et al., Protein Engin. 10, 1221-1225, 1997) or “knob into hole diabodies” (Zhu et al., Protein Sci. 6, 781-788, 1997) have been developed. However, preparation thereof is associated with considerable complexity. In addition, genetic engineering expression of a bispecific “diabody” requires a signal sequence and a ribosome binding site for each chain, which is very complicated. Although non-equimolar amounts of the variable domains may be expressed, this increases the proportion of non-functional homodimers.